Peptide compositions

ABSTRACT

Peptides have been synthesized which influence the secretion of gonadotropins by the pituitary gland of mammalians, including humans. Some of the peptides promote the release of gonadotropins, while other peptides inhibit the release.

The invention described herein was made in the course of work under agrant or award from the Department of Health and Human Services.

This is Divisional of United States Patent Application Ser. No. 509,835,now U.S. Pat. No. 4,072,668, filed Sept. 27, 1974, which is acontinuation-in-part of Ser. No. 413,314, filed Nov. 6, 1973, and nowabandoned.

The present invention relates generally to novel peptide compositionshaving influence on the release of luteinizing hormone by the pituitarygland in mammalians, including humans. More particularly, the presentinvention is directed to novel peptide compositions which enhance orinhibit the release of luteinizing hormone by the pituitary gland.

The pituitary gland is attached by a stalk to the region in the base ofthe brain known as the hypothalamus. The pituitary gland has two lobes,the anterior and posterior lobes. The posterior lobe of the pituitarygland stores and passes onto the general circulation two hormonesmanufactured in the hypothalamus, these being vasopressin and oxytocin.The anterior lobe of the pituitary gland secretes a number of hormones,which are complex protein or molecules that travel through the bloodstream to various organs and which, in turn, stimulate the secretioninto the blood stream of other hormones from the peripheral organs. Inparticular, follicle stimulating hormone and luteinizing hormone arerelease by the pituitary gland. These hormones, in combination, regulatethe functioning of the gonads to produce testosterone in the testes andprogesterone and estrogen in the ovaries, as well as regulating theproduction and maturation of gametes. These hormones are sometimesreferred to as gonadotropins or gonadotropic hormones.

The release of a hormone by the anterior lobe of the pituitary glandusually requires a prior release of another class of hormones producedby the hypothalamus. One of the hypothalamic hormones acts as a factorthat triggers the release of the gonadotropic hormones, particularlyluteinizing hormone. For convenience, luteinizing hormone is hereinafterreferred to as LH. The hypothalamic hormone which acts as a releasingfactor for LH is referred to herein as LRF wherein RF stands for"releasing factor" and the L signifies that the hormone releases LH. Asdiscussed below, LRF has been isolated and identified.

It has been demonstrated that female mammalians who have no ovulatorycycle and who show no pituitary or ovarian defect begin to secretenormal amounts of the gonadotropins, LH and FSH (follicle stimulatinghormone) after the administration of LRF. The administration of LRF issuitable for the treatment of those cases of infertility where thefunctional defect resides in the hypothalamus. Ovulation can be inducedin female mammalians by the administration of LRF. However, the dosagelevel of LRF required to influence ovulation may sometimes be high.Accordingly, it would be desirable to provide peptide materials with anenhance potency to effect the secretion of gonadotropins. It would alsobe desirable to provide peptide materials which inhibit secretion ofgonadotropins. Such inhibiting peptides can be used as contraceptives.

The principal object of the present invention is to provide peptidematerials which influence the release of gonadotropins by the pituitarygland of mammalians, including humans. Another object of the presentinvention is to provide peptide materials which have an enhanced effecton secretion of gonadotropins by the pituitary gland of mammalians,including humans. A further object of the present invention is toprovide peptide materials which have an enhanced inhibitory effect onthe secretion of gonadotropins by the pituitary gland of mammalians,including humans. Another object of the present invention is to providepeptide materials which have an enhanced effect on secretion ofgonadotropins by the pituitary gland of mammalians, including humans.

These and other objects of the present invention will become moreapparent from the following detailed description.

Generally, in accordance with the present invention, peptides have beensynthesized which influence the secretion of gonadotropins by thepituitary gland of mammalians, including humans. Some of the peptidesenhance the release of gonadotropins, while other peptides inhibit therelease when compared with known peptides which enhance or inhibitrelease of gonadotropins.

A peptide has been isolated from ovine hypothalamus fragments which hasbeen identified as being the releasing factor for the secretion of thegonadotropins. This peptide has been characterized as a decapeptidehaving the following structure:

    pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH.sub.2

For convenience, this decapeptide will be sometimes referred tohereinafter as LRF decapeptide or LRF.

Peptides are, of course, compounds which contain two or more amino acidsin which the carboxyl group of one acid is linked to the amino group ofthe other acid. The formula for LRF, as represented above, is inaccordance with conventional representation of peptides where the aminogroup appears to the left and the carboxyl group to the right. Theposition of the amino groups is identified by numbering the amino groupsfrom left to right. In the case of LRF, the hydroxyl portion of thecarboxyl group has been replaced with an amino group (NH₂). Theabbreviations for the individual amino acid groups above areconventional and are based on the trivial name of the amino acid: wherepGlu is pyroglutamic acid, His is histidine, Trp is tryptophan, Ser isserine, Tyr is tyrosine, Gly is glycine, Leu is Leucine, Arg is arginineand Pro is proline. Except for glycine, amino acids are of theL-configuration unless noted otherwise.

It has now been discovered that the substitution of D-Ala or D-Lys forGly in the 6-position of the LRF decapeptide provides a peptide materialhaving from 3 to 10 times greater potency than does LRF to effect therelease of luteinizing hormone and other gonadotropins by the pituitarygland of mammalians. The releasing effect is obtained when thesubstituted peptide is introduced into the blood stream of a mammalian.

It is known that substitution of various amino acids for His (or thedeletion of His) at the 2-position of the LRF decapeptide producespeptide materials having an inhibitory effect on the release ofluteinizing hormone and other gonadotropins by the pituitary gland ofmammalians. In particular, varying degrees of inhibition of the releaseof luteinizing hormone are obtained when His is deleted (des His) orreplaced by Asp, Cys, D-Ala, or Gly. It has been further discovered thatthe inhibitory effect of those peptides modified at the 2-position canbe greatly enhanced when D-Ala or D-Lys is substituted for Gly in the6-position of the decapeptides. For example, the peptide:pGlu-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-Gly-NH₂ is 3 times more potent as aninhibitor for the release of gonadotropins than is the same peptidewhere Gly is present in the 6-position rather than D-Ala.

The effect of the substitution of D-Ala or D-Lys in the 6-position ofthe LRF decapeptide is completely surprising. The substitution of L-Alafor Gly in the 6-position of LRF provides a peptide which has only 3percent of the potency of LRF decapeptide for effecting the release ofluteinizing hormone by the pituitary gland. The substitution of otheramino acids for Gly at the 6-position provides peptide materials withvarying levels of potency in respect to the release of luteinizinghormone and other gonadotropins but are not comparable to the use ofD-Ala. For example, the substitution of D-Val for Gly provides a peptidehaving 30 percent potency, substitution of D-Leu for Gly provides apeptide material having 60 percent potency, the substitution of D-Proprovides a peptide material having 10 percent potency. As used herein,percent potency refers to the potency compared to the LRF decapeptide.

The peptide compositions of the present invention are represented by thefollowing formula:

    pGlu-R.sub.1 -Trp-Ser-Tyr-R.sub.2 -Leu-Arg-Pro-Gly-NH.sub.2

wherein R₁ is deleted or is selected from His, Gly, Asp, Cys, des His,and D-Ala and R₂ is selected from D-Lys and D-Ala. When both D- and L-forms of an amino acid are possible, the L- form is intended unlessotherwise specifically indicated.

In general, when R₁, is Gly, or deleted (des His) the peptidecompositions are antagonistic to the secretion of gonadotropinsstimulated by LRF. When R₁ is His, the peptide compositions effectrelease of gonadotropins and are more effective than LRF for thispurpose.

Various substituted LRF peptides, as described above, have been found tohave further enhanced properties when ProNHCH₂ CH₃ is substituted forPro-Gly-NH₂ at the 9-position. For example, a potent peptide withgreater effectiveness than LRF is provided when R₁ is His, R₂ is D-Alaand Pro-NHCH₂ CH₃ is substituted for Pro-Gly-NH₂. A peptide antagonisticto secretion of gonadotropins is provided when R₁ is Gly, R₂ is D-Alaand Pro-NHCH₂ CH₃ is substituted for Pro-Gly-NH₂.

Peptides in accordance with the present invention were prepared by solidphase synthesis on a benzhydrylamine resin. The benzhydrylamine resinwas prepared in accordance with the following: ##STR1##

The resin used was a copolymer of styrene and 1 percent by weight ofdivinylbenzene.

t-Butyloxycarbonyl (BOC) protective group was used for all N^(a) aminogroups. Benzyl ethers were used for the side chain protection of Ser andTyr. All amino acids, other than D-Ala and Gly were of theL-configuration.

Coupling of a residue was carried out for 5 hours in dimethylformamide(DMF) using a 10-fold excess of BOC reagent and dicyclohexylcarbodiimide(DCC) activating reagent. The first residue is attached to thebenzhydrylamine resin by an amide bond. The coupling reaction wasmonitored by a ninhydrin test, as reported by Kaiser et al. Anal.Biochemi. 34 (1970) 595.

The deblocking procedure consisted of a 20 minute treatment in TFAcontaining 5 percent, 1,2-ethanedithiol, followed by neutralization withtriethylamine (Et₃ N) in DMF. Numerous washes with MeOH and CH₂ Cl₂followed each step.

The cleavage of the peptide from the resin and complete deprotection ofthe peptide takes place very readily at 0° C. with hydrofluoric acid(HF). Anisol was added to the resin prior to treatment with HF. Afterthe removal of HF, under vacuum, the resin was treated withethylacetate, filtered and the peptide was then eluted with acetic acidand water. The combined acetic acid-water extracts were evaporated andsubjected to purification.

Purification of the peptide was effected by partition chromatography ina gel filtration column using the elution system; n-Butanol; Aceticacid; Water (4: 1:5; volume ratio). This was followed by simple gelfiltration using 0.5 N acetic acid as eluent.

Using the above method, the peptide compositions set forth below inTables I and II were prepared. The peptide compositions were assayed invitro and in vivo. The in vitro assay was made using a five day oldprimary culture of dispersed rat pituitary cells. The LH secreted inresponse to the peptide compositions was assayed by specificradioimmunoassay for rat LH. The peptide compositions were applied tothe culture at a level of 3×10⁻⁵ M. The potency of these peptidecompositions which stimulate secretion of gonadotropins is expressed interms of percent LRF potency wherein LRF has a potency of 100. For thosepeptide compositions which are antagonistic to secretion ofgonadotropins, the culture was inoculated with 3×10⁻⁵ M of LRF and themolar ratio of the peptide required to suppress secretion of LHgenerated by the LRF was determined.

The effectiveness of the peptide compositions which stimulate secretionof gonadotropins was determined in vivo by intravenous injection of 100nanograms of the peptide into rats. The rats were chronicallyovariectomized and were treated with estrogen and progesterone prior toassay. The LH level was determined in plasma by solid phaseradioimmunoassay for rat LH. For those peptide compositions which areantagonistic to secretion of gonadotropins, the rats were injected with100 nanograms of LRF and the molar ratio of the peptide required tosuppress section of LH generated by the LRF was determined.

The results of the in vivo and in vitro tests are set forth below inTable I for those peptide compositions having greater effectiveness thanLRF. The results for those peptide compositions antagonistic tosecretion of gonadotropins stimulated by LH are set forth below in TableII.

                                      TABLE I                                     __________________________________________________________________________                                Percent LRF Potency                               Peptide Composition         In Vitro                                                                            In Vivo                                     __________________________________________________________________________    pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH.sub.2 (LRF)                                                   100%  100%                                        pGlu-His-Trp-Ser-Tyr-D-Ala-Gly-Leu-Arg-Pro-Gly-NH.sub.2 (LRF)                                             400%  500%                                        pGlu-His-Trp-Ser-Tyr-D-Lys-Leu-Arg-Pro-Gly-NH.sub.2 (LRF)                                                 500%  1,000%                                      pGlu-His-Trp-Ser-Tyr-D-Glu-Leu-Arg-Pro-Gly-NH.sub.2 (LRF)                                                 80%   30%                                         pGlu-His-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-NHCH.sub.2 CH.sub.3                                                1,500%                                                                              300%                                        __________________________________________________________________________

                                      TABLE II                                    __________________________________________________________________________                                Molar ratio necessary                                                         to produce inhibition                             Peptide Composition         In Vitro                                                                            In Vivo                                     __________________________________________________________________________    pGlu-deleted-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH.sub.2                                                     2,000 3,000                                       pGlu-deleted-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-Gly-NH.sub.2                                                   700   500                                         pGlu-Gly-Trp-Ser-Tyr-D-Lys-Leu-Arg-Pro-Gly-NH.sub.2                                                       700   500                                         pGlu-Gly-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-NHCH.sub.2 CH.sub.3                                                700   500                                         __________________________________________________________________________

It has been determined that only picogram quantities are necessary atthe pituitary gland to provide the release or the suppression of releaseof gonadotropins by the peptide compositions of the invention.Generally, administration of 500 nanograms of LRF per kilogram of bodyweight of a mammal provides a significant release of gonadotropins bythe pituitary gland. Those peptide compositions of the invention whichstimulate release of gonadotropins will produce the same response as LRFbut lower levels proportioned to their potency.

Those peptides having enhanced potency in respect to the release ofluteinizing hormone and other gonadotropins by the pituitary gland arevaluable for the treatment of mammalians reproductive disorders. Theadministration of the peptide material into the blood stream of themammalian results in the secretion of appropriate amounts of thegonadotropins. Thus, these peptide materials can be used for thetreatment of those cases of infertility where a functional defect existsin the relationship between the hypothalamus and the pituitary system.Ovulation and/or spawning can be induced in vertebrates by theadministration of the peptide materials. Substitution of D-Ala or D-Lysfor Gly in the 6-position has marked advantages, in that potency isincreased, the amount required is reduced, and oral administration ispractical.

Those peptides which have an inhibitory effect on the release ofluteinizing hormone can be used to suppress the secretion of normalamounts of the gonadotropins and can be used as contraceptives. Inaddition, they can be used to inhibit the inappropriate secretion ofgonadotropins in situations such as precocious puberty.

What is claimed is:
 1. A peptide which inhibits the release ofgonadotropins by the pituitary gland having the formula pGlu-R₁-Trp-Ser-Tyr-D-Lys-Leu-Arg-Pro-X wherein R₁ is selected from Gly, Asp,Cys, D-Ala and des R₁ and wherein X is selected from Gly-NH₂ and NHCH₂CH₃.
 2. A peptide in accordance with claim 1 wherein R₁ is Gly.
 3. Apeptide in accordance with claim 1 wherein R₁ is Asp.
 4. A peptide inaccordance with claim 1 wherein R₁ is D-Ala.
 5. A peptide in accordancewith claim 1 wherein R₁ is des R₁.
 6. A peptide in accordance with claim1 wherein X is Gly-NH₂.
 7. A peptide in accordance with claim 1 whereinX is NHCH₂ CH₃.
 8. A peptide in accordance with either claim 4 or 5wherein X is NHCH₂ CH₃.
 9. A peptide which inhibits the release ofgonadotropins by the pituitary gland having the formulapGlu-Trp-Ser-Tyr-D-Lys-Leu-Arg-Pro-Gly-NH₂.